Human erythrocytes (commonly called red blood cells or RBCs) are generated from transiently proliferating erythroblasts in marrow of mammalian adults. The inability to vastly expand erythroblasts or their precursors hematopoietic stem/progenitor cells (HSPCs) isolated from human adult peripheral blood (PB) or cord blood (CB) hinders our ability to generate ex vivo trillions of RBCs necessary for transfusion and other cell therapeutic applications. Recently, human erythroblasts and other somatic cell types were reprogrammed into induced pluripotent stem (iPS) cells that have unlimited expansion and differentiation potential. We recently developed a facile method to generate high-quality human iPS cell lines from CB or adult PB mononuclear cells (MNCs). We have used this method to generate human iPS cells from >200 adult healthy donors and patients including those with sickle cell disease, beta-thalassemia, PNH and severe aplastic anemia who need frequent and chronic blood transfusion. Moreover, we developed a feeder-free and xeno-free system to differentiate human iPS cells into definitive HSPCs and then into erythroblasts. Upon induction of terminal differentiation, these erythroblasts proliferate additional 3-4 cell divisions and form reticulocytes that express the HBB protein and exclude nuclei. Therefore, trillions of erythrocytes can be generated from a single iPS cell line that itself is expandable by cell culture For example, autologous erythrocytes could be generated from an iPS cell line derived from an alloimmunized, transfusion-dependent patient, or from donors with favorable or rare blood group antigen types. In addition, iPS cells can be genetically modified by precise genome editing technology in contrast to human HSPCs or erythroblasts that are unable to expand extensively. In this project, we aim to produce physiologic numbers of human erythrocytes ex vivo with enhanced functionality. Specifically, we propose to use selected human iPS cell lines including those with precise genetic modification for efficient and economical production of erythrocytes derived ex vivo from expandable human stem cells. The scalable ex vivo production of human erythrocytes that live longer and possess enhanced properties will provide unprecedented opportunities for transfusion medicine and other forms of cell therapies.